Method of detecting cellular immunity and application thereof to drugs

Abstract

It is intended to provide a convenient immunity monitoring system whereby T cell frequencies specific to a plural number of antigen peptides can be assayed by using a relatively small amount of blood. Peripheral monocytes are collected and frequently stimulated with an antigen without directly using any antigen presenting cells. Then T cells specific to the antigen in the thus stimulated peripheral monocytes are detected to thereby detect antigen-specific T cells. thus, diseases such as cancer can be prevented or treated with the use of a peptide having such a function, in particular, cancer tumor-rejection antigen peptide.

1 . A method for detecting antigen-specific T cells, comprising the steps of: 
 (1) collecting peripheral blood mononuclear cells;    (2) stimulating the collected peripheral blood mononuclear cells with an antigen frequently; and    (3) detecting the antigen-specific T cells in the stimulated peripheral blood mononuclear cells,    wherein the step of stimulating peripheral blood mononuclear cells in process (2) is performed without directly using antigen presenting cells.    
     
     
         2 . A method for detecting antigen-specific T cells, comprising frequent stimulation with an antigen without newly adding antigen-presenting cells to the collected peripheral blood mononuclear cells, and detection of the antigen-specific T cells in the peripheral blood mononuclear cells.  
     
     
         3 . The method of  claim 1  or  2 , wherein the peripheral blood mononuclear cells are collected from a patient with a disease.  
     
     
         4 . The method of  claim 1  wherein the patient with the disease is a patient with cancer, a virus infection or autoimmune disease.  
     
     
         5 . The method of  claim 1 , wherein the patient is HLA-A2 positive or HLA-A24 positive.  
     
     
         6 . The method of  claim 1 , wherein the antigen is an antigen protein or an antigen peptide derived from the antigen protein.  
     
     
         7 . The method of  claim 1 , wherein the protein or the antigen peptide is a tumor rejection antigen.  
     
     
         8 . The method of  claim 1 , wherein the protein or the antigen peptide is represented by any one of SEQ ID NOs: 1 to 30.  
     
     
         9 . The method of  claim 1 , wherein frequent stimulation comprises multiple stimulations at a frequency of more than twice a week.  
     
     
         10 . The method of  claim 1 , wherein frequent stimulation comprises multiple stimulations at an interval of 2 to 5 days.  
     
     
         11 . The method of  claim 1 , wherein the detection of the antigen-specific T cells is performed using the amount of cytokine produced by the T cells as an index.  
     
     
         12 . The method of  claim 1 , wherein the cytokine is IFN-γ.  
     
     
         13 . (canceled)  
     
     
         14 . The method of  claim 1 , wherein frequent stimulation is performed in the wells of a microplate.  
     
     
         15 . The method of  claim 1 , wherein the microplate is a microplate having U-shaped wells.  
     
     
         16 . The method of  claim 1 , wherein the cell count of peripheral blood mononuclear cells is 2.5×10 4  cells/well—2×10 5  cells/well using a 96-well microplate as the microplate.  
     
     
         17 . The method of  claim 1 , wherein the cell count of peripheral blood mononuclear cells is 2.5×10 4  cells/well—1×10 5  cells/well using a 384-well microplate as the microplate.  
     
     
         18 . A method for detecting an antigen that reacts with antigen-specific T cells comprising the steps of: 
 (1) collecting peripheral blood mononuclear cells;    (2) stimulating the collected peripheral blood mononuclear cells with multiple antigen candidates frequently; and    (3) determining which antigen candidates induced antigen-specific T cells,    wherein the step of stimulating the peripheral blood mononuclear cells in process (2) is performed without directly using antigen presenting cells.    
     
     
         19 . A method for detecting antigen, characterized by frequent stimulation of the collected peripheral blood mononuclear cells with an antigen without adding new antigen presenting cells, and determination of whether T cells specific to the antigen are induced in the peripheral blood mononuclear cells.  
     
     
         20 . The method of  claim 18  or  19 , wherein the peripheral blood mononuclear cells are collected from a patient with a disease.  
     
     
         21 . The method of  claim 18 , wherein the patient with the disease is a patient with cancer, a virus infection, or an autoimmune disease.  
     
     
         22 . The method of  claim 18 , wherein the patient is HLA-A2 positive or HLA-A24 positive.  
     
     
         23 . The method of  claim 18 , wherein the antigen is an antigen protein or an antigen peptide derived from the antigen protein.  
     
     
         24 . The method of  claim 17 , wherein the protein or the antigen peptide is a tumor rejection antigen.  
     
     
         25 . The method of  claim 18 , wherein the protein or the antigen peptide is represented by any one of SEQ ID NOs: 1 to 30.  
     
     
         26 . The method of  claim 18 , wherein frequent stimulation comprises multiple stimulations at a frequency of more than twice a week.  
     
     
         27 . The method of  claim 18 , wherein frequent stimulation comprises multiple stimulations at an interval of 2 to 5 days.  
     
     
         28 . The method of  claim 18 , wherein the determination of the antigen-specific T cells is performed by quantifying the amount of cytokine produced by the T cells.  
     
     
         29 . The method of  claim 18 , wherein the cytokine is IFN-γ.  
     
     
         30 . (canceled)  
     
     
         31 . The method of  claim 18 , wherein frequent stimulation is performed in the wells of a microplate.  
     
     
         32 . The method of  claim 18 , wherein the microplate is a microplate having U-shaped wells.  
     
     
         33 . The method of  claim 18 , wherein the cell count of peripheral blood mononuclear cells is 2.5×10 4  cells/well—2×10 5  cells/well using a 96-well microplate as the microplate.  
     
     
         34 . The method of  claim 18 , wherein the cell count of peripheral blood mononuclear cells is 2.5×10 4  cells/well—1×10 5  cells/well using a 384-well microplate as the microplate.  
     
     
         35 . A tailored pharmaceutical composition for treating a target disease in a patient, which is dedicated to the target disease in the patient, comprising an antigen related to the target disease in the patient.  
     
     
         36 . A tailored pharmaceutical composition for treating a target disease in a patient, which is dedicated to the target disease in the patient, comprising one or more antigens related to the target disease in the patient, wherein the antigen is selected by a method comprising the steps of: 
 (1) collecting peripheral blood mononuclear cells;    (2) stimulating the collected peripheral blood mononuclear cells with multiple antigen candidates frequently, said candidates being thought to be related to the target disease; and    (3) determining which antigen candidates induced antigen-specific T cells,    wherein the step of stimulating the peripheral blood mononuclear cells in process (2) is performed without directly using antigen presenting cells.    
     
     
         37 . A tailored pharmaceutical composition for treating a target disease in a patient, which is dedicated to the target disease in the patient, comprising an antigen selected by a method comprising stimulating collected peripheral blood mononuclear cells with an antigen frequently without adding new antigen presenting cells, and determining whether T cells specific to the antigen are induced in the peripheral blood mononuclear cells.  
     
     
         38 . (canceled)  
     
     
         39 . The tailored pharmaceutical composition of  claim 35 , wherein the peripheral blood mononuclear cells are collected from a patient with the disease.  
     
     
         40 . The tailored pharmaceutical composition of  claim 35 , wherein the patient with the disease is a patient with cancer, a virus infection or an autoimmune disease.  
     
     
         41 . The tailored pharmaceutical composition of  claim 35 , wherein the patient is HLA-A2 positive or HLA-A24 positive.  
     
     
         42 . The tailored pharmaceutical composition of  claim 35 , wherein the antigen is an antigen protein or an antigen peptide derived from the antigen protein.  
     
     
         43 . The tailored pharmaceutical composition of  claim 35 , wherein the antigen peptide is represented by any one of SEQ ID NOs: 1 to 30.  
     
     
         44 . The tailored pharmaceutical composition of  claim 35 , wherein one or more of the antigen peptides are combined.  
     
     
         45 . The tailored pharmaceutical composition of  claim 35 , wherein the disease is cancer, a virus infection, or an autoimmune disease.  
     
     
         46 . A method for treating a disease by tailored medical therapy dedicated to a target disease in a patient, comprising administering to the patient a therapeutically effective amount of an antigen related to the target disease in the patient.  
     
     
         47 . A method for treating a disease by tailored medical therapy dedicated to a target disease in a patient, comprising administering to the patient one or more antigens related to the target disease in the patient, wherein the antigens are selected by a method comprising the steps of: 
 (1) collecting peripheral blood mononuclear cells;    (2) stimulating the collected peripheral blood mononuclear cells with multiple antigen candidates frequently, said candidates being thought to be related to the target disease; and    (3) determining which antigen candidates induced antigen-specific T cells,    wherein the step of stimulating the peripheral blood mononuclear cells in process (2) is performed without directly using antigen presenting cells.    
     
     
         48 . A method for treating a disease by tailored medical therapy dedicated to a target disease in a patient, comprising administering to the patient one or more antigens selected by a method comprising stimulating collected peripheral blood mononuclear cells with an antigen frequently without adding new antigen presenting cells, and determination of whether T cells specific to the antigen are induced in the peripheral blood mononuclear cells.  
     
     
         49 . The method of any one of  claims 46  to  48 , wherein the peripheral blood mononuclear cells are collected from a patient with the disease.  
     
     
         50 . The method of  claim 46 , wherein the patient with the disease is a patient with cancer, a virus infection, or an autoimmune disease.  
     
     
         51 . The method of  claim 46 , wherein the patient is HLA-A2 positive or HLA-A24 positive.  
     
     
         52 . The method of  claim 46 , wherein the antigen is an antigen protein or an antigen peptide derived from the antigen protein.  
     
     
         53 . The method of  claim 46 , wherein the disease is cancer, a virus infection, or an autoimmune disease.  
     
     
         54 . A method for detecting allergy comprising detecting type I allergy using a non-mutating autoantigen peptide that is a tumor rejection antigen peptide recognized by HLA-A class I restricted cancer-specific cytotoxic T cells.  
     
     
         55 . A method for inhibiting allergy comprising vaccinating with a non-allergic peptide derived from an allergic peptide-retaining antigen to inhibit allergic peptide-specific IgE response and type I allergy.